A microRNA (“miRNA”) is a small, about 22 nucleotide long non-coding RNA molecule encoded by eukaryotic nuclear DNA. MicroRNA regulates gene expression transcriptionally and post-transcriptionally by base pairing with partially or fully complementary mRNA sequences which can result in translational repression of the mRNA and gene silencing or mRNA target degradation.
Duchenne muscular dystrophy is caused by loss of function mutations of the X-linked DMD gene, leading to lack of dystrophin protein at the muscle myofiber plasma membrane (Chamberlain et al., 1987; Hoffman et al., 1987a; Hoffman et al., 1988; Hoffman et al., 1987b). Becker muscular dystrophy is caused by present but abnormal dystrophin, in quality (molecular weight), or quantity (reduced percentage relative to normal muscle), or both (Beggs et al., 1991; Hoffman et al., 1989; Kesari et al., 2008). The relative amount of dystrophin protein in Becker patient muscle varies greatly, within a single muscle fiber (‘patchy’ immunostaining on subsections of myofiber membrane), between muscle fibers, and between patients. The amount of dystrophin only partially correlates with phenotype (<10% normal levels often more severe phenotypes; >10% moderate, mild, or asymptomatic phenotypes) (Hoffman et al., 1989; van den Bergen et al., 2013). The molecular underpinning for variable quantities of dystrophin include variable protein stability, variable mRNA stability, and variable mRNA translation. There is limited evidence for variable mRNA stability correlated with variable dystrophin levels (Spitali et al., 2013) as well as location of mutation and effect on expression and stability of the resulting abnormal protein (Anthony et al., 2013; Anthony et al., 2014; Anthony et al., 2011). However, series of patients with the same in-frame deletion of exons 45-47 show quite variable levels of dystrophin in their muscle, ranging from 5%-80% normal levels (Hoffman et al., 2011; Kesari et al., 2008). Patients with the same common in-frame exon 45-47 deletion mutation would be expected to show similar gene expression, similar mRNA stability, and the same mutant protein (same deleted amino acids) with expected similar stability. Thus, the molecular causes for the variable dystrophin levels in Becker patients with exon 45-47 deletions, and likely all Becker patients, have remained elusive.
Understanding the regulation of dystrophin protein quantity and stability in patient muscle is required to monitor the success of dystrophin-rescue strategies currently under development for Duchenne muscular dystrophy. Two clinical programs aimed at dystrophin rescue, stop codon read through and exon skipping antisense, have shown quite variable success, both within a specific biopsy, and between patients (Cirak et al., 2011; Finkel et al., 2013; Mendell et al., 2013). The variability was particularly evident in high dose antisense mediated exon skipping in the dystrophin-deficient dog model of DMD, where many different muscles could be systematically sampled from the same treated dog (Yokota et al., 2009; Yokota et al., 2012). The small muscle biopsy obtained from each treated patient introduces a sampling error that may not be representative of the entire patient. However, the observed variability in dystrophin expression results in challenges in interpreting success of the treatments. Perhaps more importantly, understanding the molecular basis for variable dystrophin protein expression could lead to new interventions able to improve the reliability and success of dystrophin rescue therapies.
We hypothesize that the molecular mechanisms causing variable dystrophin protein levels in Becker muscular dystrophy patients harboring the same exon 45-47 deletion might also underlie the observed variable response of muscles to exon skipping therapy. As all patients with the same exon 45-47 in-frame deletion should all share the same type of dystrophin protein, protein stability or function should not be variables in driving dystrophin protein levels. We have found that dystrophin protein levels varied between 10 unrelated BMD patients sharing the same exon 45-47 in-frame deletion, yet mRNA levels were quite similar (and similar to normal volunteer muscle). However, microRNAs targeting the dystrophin mRNA showed a high degree of variability from biopsy to biopsy, and the levels and numbers of these microRNAs correlated well with dystrophin protein levels. Our data suggests that variable expression of microRNAs in patient muscle may drive observed variability in dystrophin protein levels in both BMD patients, and DMD patients treated with exon-skipping antisense therapy.
As described herein, the inventors identified the cause of variable dystrophin levels to be binding of specific microRNAs to the dystrophin mRNA 3′ untranslated region (3′ UTR). This enables new methods for treating dystrophin-related conditions, disorders, or diseases, such as Becker muscular dystrophy, by inhibiting these kinds of miRNAs to increase expression of dystrophin protein in cells, especially in patient muscle cells. The blocking of the specific microRNAs which inhibit dystrophin expression increases the translation of mRNA encoding dystrophin protein.
Novel compounds and pharmaceutical compositions, certain of which have been found to modulate dystrophin expression have been discovered, together with methods of synthesizing and using the compounds including methods for the treatment of dystrophin-mediated diseases in a patient by administering the compounds.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative thereof that binds to at least one of miR-146a, miR-146b-5p, miR-223, miR-320a, miR-374a, or miR-382 or to portions thereof, and that increases expression of dystrophin protein.
Certain compounds disclosed herein may possess useful dystrophin modulating activity, and may be used in the treatment or prophylaxis of a disease or condition in which dystrophin plays an active role. Thus, in broad aspect, certain embodiments also provide pharmaceutical compositions comprising one or more compounds disclosed herein together with a pharmaceutically acceptable carrier, as well as methods of making and using the compounds and compositions. Certain embodiments provide methods for modulating dystrophin. Other embodiments provide methods for treating a dystrophin-mediated disorder in a patient in need of such treatment, comprising administering to said patient a therapeutically effective amount of a compound or composition according to the present invention. Also provided is the use of certain compounds disclosed herein for use in the manufacture of a medicament for the treatment of a disease or condition ameliorated by the modulation dystrophin.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that is partially complementary to at least one of miR-146a, miR-146b-5p, miR-223, miR-320a, miR-374a, or miR-382.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that binds to the seed sequence of at least one of miR-146a, miR-146b-5p, miR-223, miR-320a, miR-374a, or miR-382, wherein said seed sequence binds to the 3′UTR of dsytrophin mRNA.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that comprises a sequence that is fully complementary to the seed sequence of at least one of miR-146a, miR-146b-5p, miR-223, miR-320a, miR-374a, or miR-382.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to a full complement of a seed sequence of at least one of miR-146a, miR-146b-5p, miR-223, miR-320a, miR-374a, or miR-382.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that comprises a sequence that is fully complementary to at least one of miR-146a, miR-146b-5p, miR-223, miR-320a, miR-374a, or miR-382.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to a full complement of at least one of miR-146a, miR-146b-5p, miR-223, miR-320a, miR-374a, or miR-382.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that comprises a sequence that is fully complementary to the sequence of at least one of miR-146a, miR-146b-5p, miR-223, miR-320a, miR-374a, or miR-382, except that it contains 1, 2, 3, 4, 5, 6, or 7 deletions, substitutions, or insertions to said fully complementary sequence.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that comprises polynucleotide sequences complementary to at least two of miR-146a, miR-146b-5p, miR-223, miR-320a, miR-374a, or miR-382.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that binds to miRNA and sequesters it inside of a muscle cell or other kind of cell.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that increases the expression of dystrophin protein in muscle cells.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that increases the expression of dystrophin protein in muscle cells.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that binds to miR-146a.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that binds to miR-146b-5p.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that binds to miR-223.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that binds to miR-320a.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that binds to miR-374a.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative that binds to miR-382.
In certain embodiments of the present invention disclosed herein is a pharmaceutical composition comprising a nucleic acid molecule or derivative as disclosed herein and a pharmaceutically acceptable carrier or excipient.
In certain embodiments of the present invention disclosed herein is a covalent or non-covalent conjugate comprising the nucleic acid molecule or derivative as disclosed herein and a second targeting effector moiety.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative thereof able to functionally inactivate the action of miR-146a, miRNA-146b-5p, miR-223, miR-320a, miR374a, and/or miR-382 and increase dystrophin protein translation in muscle.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative thereof that binds to at least one miRNA selected from the group consisting of miR-146a, miRNA-146b-5p, miR-223, miR-320a, miR374a, and miR-382 by sequence complementarity and that selectively sequesters said miRNA in muscle cells.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative thereof, comprising one or more protector sequences complementary to miR-146a, miRNA-146b-5p, miR-223, miR-320a, miR374a, and/or miR-382 sequences on the 3′UTR of dystrophin; wherein the 3′UTR seed sequences of dystrophin bound by these miRNAs are selected from the group consisting of:
a. miR-146a:(SEQ ID NO: 16)5′-UGAUUGUUCAUAAUACAUAAAGUUCUCUGUAAUUACAACUAAAUUAU-3′ DMD b. miR-146b:(SEQ ID NO: 17)5′-AUGAUUGUUCAUAAUACAUAAAGUUCUCUGUAAUUACAACUAAAUUA-3′ DMD c. miR-223:(SEQ ID NO: 18)5′-AAGUAUAUAAAUACUAUAGUUAUAUAGAUAAAGAGAU-3′DMD d. miR-320a:(SEQ ID NO: 19)5′-CAGGUACUGAGUUCUUACUUGAGUAUCAUAAUAU-3′DMD e. miR-374a Site 1:(SEQ ID NO: 20)5′-UUUGUGAAGGGUAGUGGUAUUAUACUGUAGAUU-3′ DMD f. miR-374a Site 2:(SEQ ID NO: 21)5′-AAUACACAGGACUUAUUAUAUCAGAGU-3′ DMD g. miR-374a Site 3:(SEQ ID NO: 22)5′-CCAAAUAUAUGCCUUACUAUUGUAUUAUAGUACUGCU-3′DMD h. miR-382:(SEQ ID NO: 23)5′-AGCUCCAGAUGUUUCUCAUUUUAAACAACUUUCCACUGACAACGAAA-3′ DMDand c. miR-223:(SEQ ID NO: 18)5′-AAGUAUAUAAAUACUAUAGUUAUAUAGAUAAAGAGAU-3′ DMD.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative thereof wherein at least one of the protector sequences for the group consisting of:
a. miR-146a:(SEQ ID NO: 24)5′-ACAGAGAACTTTATGTATTATGAAC-3′ b. miR-146b:(SEQ ID NO: 25)5′-ACAGAGAACTTTATGTATTATGAAC-3′ c. miR-320a:(SEQ ID NO: 26)5′-ATGATACTCAAGTAAGAACTCAGTA-3′ d. miR-374a Site 1:(SEQ ID NO: 27)5′-ACAGTATAATACCACTACCCTTCAC-3′ e. miR-374a Site 2:(SEQ ID NO: 28)5′-CTGATATAATAAGTCCTGTGTATTC-3′ f. miR-374a Site 3:(SEQ ID NO: 29)5′-CAGTACTATAATACAATAGTAAGGC-3′and g. miR-382:(SEQ ID NO: 30)5′-CGTTGTCAGTGGAAAGTTGTTTAAA-3′ DMD
or a protector sequence having 1, 2, 3, or 4 insertions, substitution, or deletions to said sequences.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative thereof that comprises two, three, four or more of said protector sequences that are complementary to said mRNAs.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative thereof wherein at least one nucleotide is not complementary to the corresponding nucleotide comprised in the region from nt 9 to nt 14 of SEQ ID NOs:16-23.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative thereof wherein at least three nucleotides are not complementary to the corresponding nucleotide comprised in the region from nt 9 to nt 14 of SEQ ID NOs:16-23.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative thereof that is able to compete with at least one miRNA selected from the group consisting of miR-146a, miRNA-146b, miR-223, miR-320a, miR374a, and miR-382 for binding to the 3′UTR dystrophin mRNA.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative thereof comprising a sequence that is complementary to the 3′UTR dystrophin mRNA region which is recognized by a miRNA selected from the group consisting of miR-146a, miRNA-146b, miR-223, miR-320a, miR374a, and/or miR-382 sequences SEQ ID NOs:16-23.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative thereof being a modified synthetic oligonucleotide.
In certain embodiments of the present invention disclosed herein is a nucleic acid molecule or derivative thereof belonging to the group of locked nucleic acids, methylated oligonucleotides, phosphoro-thiolated oligonucleotides, morpholino oligonucleotides, and poly-morpholino oligonucleotides.
In certain embodiments of the present invention disclosed herein is a method for increasing the translation of dystrophin in a patient in need thereof, comprising the administration of a therapeutically active amount of a nucleic acid molecule or derivative thereof as disclosed herein.
In certain embodiments of the present invention disclosed herein is a method for modulating the expression of dystrophin comprising contacting a cell expressing mRNA encoding dystrophin with the nucleic acid or derivative as disclosed herein.
In certain embodiments of the present invention disclosed herein is a method as disclosed herein further comprising administering the nucleic acid or derivative as disclosed herein to a subject having a dystrophin-related disease in an amount effective to increase the expression of dystrophin.
In certain embodiments of the present invention disclosed herein is a method as disclosed herein, wherein said dystrophin-related disease is muscular dystrophy.
In certain embodiments of the present invention disclosed herein is a method as disclosed herein, wherein said dystrophin-related disease is Becker muscular dystrophy.
In certain embodiments of the present invention disclosed herein is a method as disclosed herein, wherein said dystrophin-related disease is Duchenne muscular dystrophy.
In certain embodiments of the present invention disclosed herein is a method as disclosed herein, further comprising the treatment of said patient with a codon read through antisense therapy.
In certain embodiments of the present invention disclosed herein is a method as disclosed herein, further comprising the treatment of said patient with an exon skipping antisense therapy.
In certain embodiments of the present invention disclosed herein is a method as disclosed herein, further comprising the administration of VBP-15.
In certain embodiments of the present invention disclosed herein is the use of a therapeutically active amount of a nucleic acid molecule or derivative thereof as disclosed herein for increasing the translation of dystrophin in a patient in need thereof.
In certain embodiments of the present invention disclosed herein is the use of a nucleic acid or derivative as disclosed herein for modulating the expression of dystrophin comprising contacting a cell expressing mRNA.
In certain embodiments of the present invention disclosed herein is the use of a nucleic acid or derivative as disclosed herein in an amount effective to increase the expression of dystrophin.
In certain embodiments of the present invention disclosed herein is a use as disclosed herein, wherein said dystrophin-related disease is muscular dystrophy.
In certain embodiments of the present invention disclosed herein is a use as disclosed herein, wherein said dystrophin-related disease is Becker muscular dystrophy.
In certain embodiments of the present invention disclosed herein is a use as disclosed herein, wherein said dystrophin-related disease is Duchenne muscular dystrophy.
In certain embodiments of the present invention disclosed herein is a use as disclosed herein, further comprising the treatment of said patient with a codon read through antisense therapy.
In certain embodiments of the present invention disclosed herein is a use as disclosed herein, further comprising the treatment of said patient with an exon skipping antisense therapy.
In certain embodiments of the present invention disclosed herein is a use as disclosed herein, further comprising the administration of VBP-15.